Alpha-granules, the major secretory organelle of platelets, contain hundreds of proteins that are released upon activation. Interestingly, many of the stored proteins have seemingly opposite function, such as those with pro- or anti-angiogenic properties. A major unanswered question in platelet physiology is: Are platelets an active participant specifically releasing context-appropriate material from their -granules or are they random delivery devises? Here we address important aspects of that central question through three Specific Aims. Specific Aim 1: To test the hypothesis that human platelets contain a single major -granule population in which individual cargo proteins are packaged into distinct zones. Through the combined application of electron tomography, immunogold labeling, and super-resolution light microscopy, we will analyze a whole platelet both with respect to granule structure and protein distribution. Using these structural approaches, we will determine the extent of homogeneity, or heterogeneity, in structure and cargo protein distribution in the human -granule population. Specific Aim 2: To test the hypothesis that specialized -granule subdomains/extensions provide a spatial basis for differential membrane fusion/protein secretion to the plasma membrane/OCS in response to agonists. In this Aim, we apply the imaging approaches, from Aim 1, to characterize the structural basis on which platelet -granule secretion can support differential protein release. Our data and that of others suggest that differential release is a normal outcome of -granule secretion. To date, our Preliminary Data are consistent with a model in which important fusion machinery proteins such as the v- SNARE, VAMP-8, are concentrated over distinct subdomains of the -granule and hence may mediate subdomain specific fusion. Mouse platelets from gene knockouts will be facilitate experiments designed to reveal the accumulation of intermediates in granule release. Specific Aim 3: To test the hypothesis that VWF and/or cytoskeletal elements provides an organizing principle for platelet -granule structure and function. Reversible depolymerization of granule VWF has the therapeutic potential to modulate -granule secretion through affecting protein zoning and granule shape. The proposed research is both significant and innovative. Our overarching hypothesis of a granule organized structurally into specific subdomains designed for agonist-responsive secretion provides an innovative intellectual framework that drives experiments towards incisive answers. This framework can lead to revealing answers that would not come otherwise. Our experience in high-resolution imaging technology brings a novel toolset to the platelet field needed to definitively answer the central question raised. Our work will provide a reference framework for future therapeutic design.